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The main aim of this master thesis was the simultaneous detection of four selected plant viruses ? Apple mosaic virus, Plum pox virus, Prunus necrotic ringspot virus and Prune harf virus, by microarrays. The intermediate step in the process of the detection was optimizing of multiplex polymerase chain reaction (PCR).

Le virus des nervures jaunes et nécrotiques de la betterave (Beet necrotic yellow vein virus, BNYVV) est l’agent infectieux responsable de la rhizomanie de la betterave sucrière, une maladie caractérisée par une prolifération anarchique du chevelu racinaire. Le Beet soil-borne mosaic virus (BSBMV) appartient également au genre Benyvirus mais n’est retrouvé qu’en Amérique du Nord. Ce virus, identifié pour la première fois au Texas, est morphologiquement et génétiquement semblable au BNYVV mais sérologiquement éloigné. Compte tenu des différences moléculaires existant, le BSBMV et BNYVV correspondent à deux espèces virales distinctes. Mon projet de thèse a consisté à étudier les interactions moléculaires entre le BNYVV et le BSBMV et rechercher les mécanismes impliqués dans la pathogénicité de ces deux virus. Des clones complets cDNA infectieux du BNYVV étaient disponibles, tout comme ceux de BSBMV. Compte tenu de l’aspect versatile de l’obtention de transcrits infectieux de ces différents clones, j’ai entrepris de produire des clones cDNA de chacun des ARN viraux sous contrôle d’un promoteur constitutive végétal pour initier l’infection par agroinfiltration. Les plantes hôtes Chenopodium quinoa et Nicotiana benthamiana ont été inoculées par des transcrits et agroinfiltrées pour initier l’infection virale et étudier l’interaction entre les ARN génomiques 1 et 2 des deux virus et étudier les propriétés de constructions chimères. En parallèle à ce travail, j’ai réalisé la caractérisation du suppresseur de RNA silencing du BSBMV en le comparant à celui du BNYVV. / The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.

Clostridium perfringens causa enterite necrótica aviária devido à produção de toxinas que lesam o intestino. Neste estudo, de 94 amostras nove apresentaram C. perfringens, totalizando 22 isolados. Todos exceto um isolado, possuíram os genes nanI (95%) e/ou nanJ (81%), e 19/22, mostraram atividade neuraminidase em hemácias de frango. A atividade hemaglutinante foi observada em poucos isolados (26%). Todos os isolados foram plc positivos (toxina α), sendo classificados como tipo A. Sete isolados (31,8%) abrigaram o gene tpeL que codifica a toxina TpeL. Isolados tpeL+ mostraram efeito citotóxico característico da ação desta toxina. Alguns isolados mostraram capacidade de aderir e invadir células Vero. A maioria dos isolados foi resistente à sulfaquinoxalina (100%), cefalexina (95%) e eritromicina (95%) e sensíveis (100%) à cefoxitina, amoxicilina, enrofloxacina, amoxicilina-ácido clavulânico, penicilina-estreptomicina, cloranfenicol e metronidazol. Todos os isolados foram agrupados geneticamente em sete clusters, apresentando se como um grupo heterogêneo. / Clostridium perfringens cause avian necrotic enteritis due to production of toxins that damage the intestine. In this study, nine out of 94 samples had C. perfringens, totaling 22 isolates. All the isolates with exception of one, possessed the genes nanI (95 %) and/or nanJ (81 %), and 19/22 showed neuraminidase activity in chicken erythrocytes. The hemagglutinating activity was observed in a few isolates (26 %). All isolates were plc positive (toxin α) being classified as type A. Seven isolates (31.8%) harbored tpeL gene encoding the toxin TpeL. TpeL + isolates showed characteristic cytotoxic effect of the action of this toxin. Some isolates showed ability to adhere and invade Hep-2 cells. Most of the isolates were resistant sulphaquinoxaline (100%), cephalexin (95%) and erythromycin (95%) and sensitivity (100%) to cefoxitin, amoxicillin, enrofloxacin, amoxicillin - clavulanic acid, penicillin -streptomycin, chloramphenicol and metronidazole. All isolates were genetically grouped into seven clusters, presenting itself as a heterogeneous group.

Made available in DSpace on 2014-08-20T13:32:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-10-02 / Avian Necrotic Enteritis (NE) is an acute enterotoxaemia caused by Clostridium perfringens A and C. The control of the disease is based on antibiotics added to animal feed. The ban of this practice by consumer markets, considered the biggest challenge to industrial aviculture, demanded the adoption of other alternatives for its control, among others, immunization with recombinant vaccines. The aim of this work was to produce and evaluate C. perfringens recombinant α toxin (rAT) vaccines adjuvanted with either Al(OH)3 (rAT+Al(OH)3 or recombinant B subunit of the heat-labile enterotoxin of Escherichia coli (rLTB) (rAT+rLTB), and a chimeric protein containing the α toxin fused to rLTB (rLTB-AT). The rAT+Al(OH)3 was innocuous and protected mice against a challenge with native α toxin (sT), and it was immunogenic and did not affect productivity parameters in broilers. The rAT+rLTB showed a dose-protection relationship in mice, while rLTB-AT did not protect mice against sT challenge. The rAT could be an alternative for controlling NE. / A Enterite Necrótica Aviar (ENA) é uma enterotoxemia aguda, causada pelos Clostridium perfringens A e C, cujo controle baseia-se na adição de antibióticos na ração. A restrição dessa prática pelo mercado consumidor, que tornou seu controle o maior desafio para o setor avícola, exigiu a adoção de novas estratégias para o controle, entre elas a imunização. Vacinas recombinantes vêm despertando grande interesse entre pesquisadores e empresas do setor. O objetivo deste trabalho foi elaborar vacinas de toxina α recombinante de C. perfringens (rAT) utilizando como adjuvantes Al(OH)3 (rAT+Al(OH)3) e subunidade B recombinante da enterotoxina termolábil de Escherichia coli (rLTB) (rAT+rLTB), e construir e avaliar uma proteína quimérica contendo rAT fusionada a rLTB (rLTB-AT). A rAT+Al(OH)3 foi inócua e protetora contra agressão de toxina α nativa (sT) em camundongos, e imunogênica em frangos de corte, sem afetar a produtividade. A rAT+rLTB demonstrou relação dose-proteção em camundongos, entanto a rLTB-AT não protegeu camundongos contra agressão de sT. A rAT demonstrou ser uma alternativa para controlar a ENA. Palavras-chave: Enterite Necrótica Aviar, Clostridium perfringens A, toxina α recombinante, vacinas.

L’entérite nécrotique aviaire causée par Clostridium perfringens est une maladie économiquement dévastatrice et celle-ci est en émergence dans les troupeaux de poulets de chair éliminant l’usage des antibiotiques. À ce jour, aucune alternative en élevage ne permet de prévenir efficacement la maladie et un contrôle par une stratégie vaccinale serait des plus prisé. Une approche par génomique comparative jumelée à la vaccinologie réverse soustractive et comparative identifiant des protéines bactériennes de surface immunogènes figure parmi les approches méthodologiques des plus prometteuses pour le développement rapide d’un vaccin efficace. Une étude génomique comparative réalisée sur 48 souches de C. perfringens provenant de poulets de chair en santé ou affectés par l’entérite nécrotique a permis d’établir que les génomes analysés étaient composés de 155 700 protéines distinctes, où 13% étaient extracellulaires, 65% cytoplasmiques et 22% membranaires. L’évaluation du pouvoir immunogène de ces protéines à l’aide de l’outil de prédiction VaxiJen v.2.0 a permis d’identifier 4 catégories de scores pour les protéines identifiées, allant de 0,5 (seuil minimal recommandé) à 1,5. Les protéines présentant les scores les plus élevés ont été majoritairement associées à des localisations extracellulaires. La combinaison du score d’immunogénicité et de la localisation cellulaire des protéines analysées a mené à la sélection de 12 protéines candidates vaccinales, la plupart d’entre elles étant de fonction hypothétique. Une description plus approfondie de ces protéines permettra de mieux définir leur fonction, d’évaluer leur potentiel antigénique réel en caractérisant leur interaction avec le système immunitaire de la volaille et ultimement, d’évaluer leur rôle probable dans la pathogénie de l’entérite nécrotique. / Avian necrotic enteritis caused by Clostridium perfringens is a disease with a major economical impact, generating losses up to 6 billion dollars for the poultry industry worldwide. This disease appears in broiler chicken flocks that no longer employ the use of antibiotics. To date, no alternative method allows for the efficient prevention of necrotic enteritis (NE) and a control by a vaccinal strategy would be mostly prized. A comparative genomics approach as well as comparative and subtractive reverse vaccinology identifying immunogenic bacterial surface proteins is one of the most promising methodologies for the rapid development of an efficient vaccine. A comparative genomic study was performed on 48 C. perfringens strains isolated from healthy broiler chickens and from broilers affected by necrotic enteritis. From this study, it was established that the genomes analyzed were composed of 155 700 distinct proteins where 13% were predicted to have an extracellular expression, 65% at the cytoplasma level and 22% within the plasma membrane. The evaluation of the immunogenic potential of these proteins was established with the prediction software VaxiJen v2.0 for which a 0.5 threshold score allowed for the identification of four score categories among the identified proteins, from 0.5 to 1.5. For the most part, proteins with the highest scores were associated with an extracellular localisation. The combination of the immunogenicity score and localisation of the analysed proteins led to the selection of 12 vaccinal candidate proteins that were mostly identified as hypothetical. A more in-depth description of these proteins would allow the assessment of their function, the evaluation of their true immunogenic potential by characterizing their interaction with the avian immune system and ultimately, evaluate their probable role in the pathogenesis of necrotic enteritis.

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vegetable crops. Micro-Tom is a model tomato cultivar used for research due to its small size and short time to fruiting. This project evaluated I. walleriana and Micro-Tom transformed with Agrobacterium. The construct contained GFP (green fluorescent protein) and hygromycin antibiotic-resistant selectable markers, and the antisense sequence of open reading frame of INSV nucleocapsid protein (N). The N gene is expected to confer INSV resistance by RNA interference or gene silencing. The presence of transgenes was confirmed by PCR. Transgenic Impatiens was selfed for two generations. Transgenic Micro-Tom was selfed for 4 generations. Spinach was used as an INSV reservoir. Impatiens, spinach and Micro-Tom were mechanically inoculated with INSV and evaluated visually, with assay tests, ELISA testing, and PCR. Spinach was successfully infected with INSV six times of seven attempts. Impatiens and Micro-Tom had no successful inoculations of three and five attempts, respectively. / Master of Science

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